<% vol = 16 number = 2 nextlink = 87 prevlink = 81 titolo = "THE SENSITIVITY TO HONEY OF MULTIDRUG-RESISTANT PSEUDOMONAS AERUGINOSA FROM INFECTED BURNS" volromano = "XVI" data_pubblicazione = "June 2003" header titolo %>

Subrahmanyam M.1, Hemmady A.R.2, Pawar S.G.2

Department of Surgery1 and Microbiology,2 Government Medical College, Miraj and General Hospital,Sangli, Maharashtra, India


SUMMARY. A total number of 44 strains of Pseudomonas aeruginosa isolated from infected burns showing multidrug resistance were studied for sensitivity to honey on the principle of minimum inhibitory concentration (MIC). All strains showed similar sensitivity to honey with MIC at 25% concentration, 39 strains at 20%, 15 at 15%, and three at 10%. The present study showed that strains of Pseudomonas aeruginosa resistant to routinely used and higher antibiotics were sensitive to the antibacterial action of honey. In the present era of multiple drug-resistant organisms, honey can be used as an effective, cheap, easily available, non-toxic, extremely useful, and reliable adjuvant without any adverse side effects in the treatment of burn wound infection.


Introduction

Despite recent advances in antimicrobial therapy, several types of wounds - burn wounds, chronic non-healing ulcers, post-traumatic wounds, etc. - still show resistance to routine wound treatment.

To reduce bacterial growth under the burn surface, various agents such as silver sulphadiazine, 5% silver nitrate, and Sulfamylon have been used and various methods of grafting have been tried. However, none of these methods has effectively eliminated the problem of infection.1 The emergence of antibiotic-resistant pathogens makes it essential to look for an alternative to counter these multidrug-resistant organisms. Pseudomonas aeruginosa is detected in 4% of wounds in hospital patients and it is generally accepted to be an important pathogen in chronic wounds and burns.

It has been shown that these organisms develop more readily in extensive burns than in minor burns.2

They are resistant to commonly used and the higher antibiotics and a search for a more effective inhibitory agent is imperative.

Honey has been suggested in order to enhance the healing of wounds and pressure sores. Honey’s antibacterial and antifungal properties have been well documented.3 Honey is produced from many floral sources and its antibacterial activity varies, which explains why there is so much variation in in vitro testing of the sensitivity of wound-infecting bacteria to honey. In studies conducted on superficial, partial-thickness, and deep burns, it has been observed that honey also has a mopping-up effect on free radicals, which leads to reduced scarring and contractures. Honey probably plays a role in the initial phase by limiting lipid peroxidation and at a later stage by controlling infection and promoting healthy granulation.4,5 In multidrug-resistant Pseudomonas infection, honey may have a role to play, and the present study aims to investigate the efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected burn wounds.

Material and methods

Strains of Pseudomonas aeruginosa were isolated from swabs collected from burn patients in General Hospital, Sangli, Maharashtra, India. The identity of all the strains was confirmed.

The antibiotic sensitivity of the organisms was performed on Muller Hinton agar plates by the Kirby-Bauser disk diffusion method.

The organisms were then subcultured on a nutrient agar slant.

Selection of honey. Honey obtained from five plant sources was assessed for antibacterial activity as described by White et al.,6 using Staphylococcus aureus. A fifty per cent honey solution was prepared, in order to produce media with 25, 20, 15, 10 and 5% of honey. These solutions were poured into Petri dishes and after solidification they were inoculated with a drop of 24-hour-old culture diluted to 1/1000 in a broth of S. aureus. Observations were made after 24 h incubation at 37 °C. On the basis of the inhibition, in 5 to 25% media, gradations were given respectively from 5 to 0. The honey with the highest gradation and the highest antibacterial activity (honey from Apis Cerena of the plant source Syzygium cumini) was selected for this study. It has a pH of 3.0, water content of 20%, and a specific gravity of 1.37 at 27 °C.

Sterility of honey. The sterility of the honey was tested by inoculating a loopful of the undiluted and unprocessed honey from the source, as described above, on blood agar, MacConkey agar, and Sabouraud’s agar with the standard inoculating loop of nichrome wire. The plates of blood agar, MacConkey agar, and one slant of Sabouraud’s agar were incubated at 37 °C and another Sabouraud’s agar slant was kept at room temperature. No organism growth was observed on blood agar and MacConkey agar even after 48 h, and cultures on Sabouraud’s agar were sterile for up to three weeks. The sterility of the honey used in the study was thus confirmed.7

Different concentrations of honey (vol/vol) were prepared in MH medium at 56 °C in order to give its final concentrations of 5, 10, 15, 20, 25, and 30%. The minimum inhibitory concentration (MIC) of the honey was determined as described earlier.8 Clinical isolates were incubated at 37 °C for 24 h and observed for growth or no growth. MIC was recorded as the lowest concentration of honey preventing growth of each isolate. Isolates inoculated only on MH medium without honey served as control.

Results

All 44 isolates exhibited a similar antibiogram. A maximum number of four strains showed sensitivity to antibiotics commonly used in our hospital (Table I). These organisms were therefore considered to be multidrug-resistant.

<% createTable "Table I ","Number of strains of organisms sensitive to antibiotics",";Strains (no);A.;C.;E.;K.;P.;S.;T.;G.;Amox.;Cip.;Nor.@;Sensitive;1;0;0;1;0;2;2;2;1;4;2@;Resistant;43;44;44;43;44;42;42;42;43;40;42@;Total;44;44;44;44;44;44;44;44;44;44;44@§1,12§A. = ampicillin C. = chloramphenicol E. = erythromycin K. = kanamycin P. = penicillin S. = streptomycin T.= tetracycline G. = gentamicin Amox. = amoxicillin Cip. = ciprofloxacin Nor. = norfloxacin","",4,300,true %>

The MIC for the honey used in this study is shown in Table II. Out of the 44 strains, 100% inhibition was observed at honey concentrations of 25 and 30%, compared with 88.6% at 20%, 34.1% at 15%, and 6.8% at 10%.

Thus all strains were inhibited at 25% honey concentration (mean MIC value, 28.6%).

<% createTable "Table II ","Minimum inhibitory concentrations (percentage vol/vol)",";Organism§1,6§Number and percentage of strains inhibited on Muller-Hinton agar with honey at various concentrations@;Pseudomonas aeruginosa;5%;10%;15%;20%;25%;30%@; 0;3
(6.8%);15
(34.1%);39
(88.6%);44
(100%);44
(100%)","",4,300,true %>

Discussion

Pseudomonas aeruginosa can cause sepsis, pneumonia, and infection in burned skin. It occupies multiple ecological niches in nature by virtue of its minimal growth requirements and its ability to produce a large number of extracellular protective and toxic substances. Some of these substances, such as slime glycolipoprotein, haemolysis, fibrinolysis, lecithinase, elastase, Dnase, and phospholipase, may contribute to the pathogenicity of this opportunistic micro-organism.9 Their occurrence in burns and other wounds continues to contribute to extended treatment times, to increased costs and delayed healing rates, causing skin grafts to fail, and to increased risks of septicaemia. They are multidrug-resistant to the most commonly used antiseptics, disinfectants, and antibiotics, and the search for new antimicrobial agents continues.

Honey has been used an adjuvant method for accelerating wound healing since ancient times, particularly in the treatment of burns. When used as a wound dressing, 15-30 ml of honey can be applied directly onto the

burn wound, or soaked in gauze before application. Occlusive or absorbent secondary dressings are applied to prevent the honey from oozing out, and the frequency of dressing changes depends on how rapidly the honey is diluted by the exudate, which declines as treatment progresses.

Several prospective randomized controlled clinical trials conducted in superficial and partial-thickness burns have led to effective control of clinical infection that is much better than that achieved with silver sulphadiazine and other substances.4,5 A survey conducted by Hermans10 in 1998 to review worldwide treatment of burns found that honey was used in 5.5% of instances, while silver sulphadiazine 1% was the preferred treatment for partial-thickness and mixed burns.

Subsequent to this review, a number of clinical trials4,5 have been published that have reported good results with honey owing to its anti-inflammatory, antibacterial, and anti-oxidant effect.

Different values have been reported for the sensitivity of Pseudomonas to the antibacterial activity of honey. The MIC against Pseudomonas ranged from 3 to 30% (vol/vol).12 In this study the MIC was 25% (vol/vol) for all strains, which were obtained from infected burn wounds, and these were more useful for treating clinical infections. The floral origin of honey and its antibacterial potency have been mentioned as factors accounting for the variations in MIC reported in various studies.11 However, it was observed in clinical trials conducted by the present author that the floral origin of honey and its antibacterial potency appeared to make no difference to the effectiveness of honey in the treatment of burns. It was observed that honey treatment led to a reduction in inflammation, scarring, and contractures, and the antioxidant effect of honey in the mopping-up free radicals, together with its antibacterial action, low pH, high viscosity and hygroscopic effect, etc., all seem to be play a combined role in the efficacy of honey in burns treatment.

Conclusions

Honey is cheap, cost-effective, and easily available, which makes it a favourite medicine for several ailments as well as a dressing for burns and other wounds. As shown in this study, standard tests proved honey to be sterile and so far no clinical study has shown any clostridial infection after its use in wounds, and honey can thus be safely used in an unprocessed and undiluted condition, without any risk. For this reason it can be used in the underdeveloped and developing world where facilities for sterilizing honey by gamma irradiation may not be available. In this way a cheap and effective medicine will not be denied to the people who need it.


RESUME. Les Auteurs ont étudié 44 souches de Pseudomonas aeruginosa isolées dans des brûlures infectées qui présentaient une résistance multiple aux médicaments afin d’établir la sensibilité au miel sur le principe de la concentration inhibitoire minimale (CIM). Toutes les souches démontraient une sensibilité semblable au miel avec une CIM de 25% concentration, 39 souches à 20%, 15 à 15%, et trois à 10%. Les Auteurs démontrent que les souches étudiées de Pseudomonas aeruginosa résistantes aux antibiotiques supérieurs normalement utilisés étaient sensibles à l’action antibactérienne du miel. Devant la présence dans notre ère d’un grand numéro d’organismes qui présentent une résistance multiple aux médicaments, le miel peut être utilisé comme un support efficace, économique, facilement disponible, non-toxique, extrêmement utile et fiable sans aucun effet indésirable dans le traitement des infections des brûlures.


Bibliography

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  9. Torregrossa M.V., Valentino L., Cucchiara P. et al.: Prevention of hospital-acquired infections in the Palermo burn centre. Ann. Burns and Fire Disasters, 13: 143-7, 2000.
  10. Hermans M.H.E.: Results of a survey on the use of different treatment options for partial- and full-thickness burns. Burns, 24: 539-51, 1998.
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<% riquadro "This paper was received on 9 April 2003.

Address correspondence to: Dr M. Subrahmanyam, Old Civil Hospital Compound, Rajwada Chowk, Sangli 416416, Maharashtra, India.
E-mail: san_avanism@sancharnet.in" %>

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